JSH-23, an NF-κB inhibitor（IC50= 7.1 μM）, suppresses the transcriptional activity.

Within diabetic rat models, JSH-23 (3 mg/kg) effectively reduces neuroinflammation and enhances antioxidant defenses, significantly reversing neural conduction impairments and ultimately ameliorating insufficient neurovascular perfusion.

In RAW 264.7 cells, JSH-23 at concentrations below 100 μM did not exhibit significant cytotoxic effects and was capable of inhibiting lipopolysaccharide-(LPS)-induced apoptotic chromatin condensation. Furthermore, in primary cerebellar neurons of mice, JSH-23 inhibited the LPS-induced nuclear translocation of NF-κB p65 without affecting the degradation of IκBα.

Measurement of NF-κB transcriptional activity: Macrophages RAW 264.7 transfected stably with reporter plasmid of pNF-κB-SEAP-NPT are treated with 1 μg/ml LPS and/or sample for 16 hours. As the reporter, SEAP activity in the cell-free culture media is measured as followed. Single cell-derived stable transfectants are plated in 5 ml of T-25 flask, and the media is decanted 24 h later. At this time, cells are washed twice with phosphate-buffered saline, and incubations are initiated by addition of new media. Chemicals are added to the culture medium after 24 h of incubations. Aliquots (25 ml) of medium from a control or chemical-treated cultures are taken at 0, 3, 20, 24, 48, and 72 h, heated at 65°C for 5 min to eliminate the alkaline phosphatase activity, and used immediately or stored at -20°C. Mixtures consisting of dilution buffer (25 ml), assay buffer (97 ml), culture media (25 ml), and 4-methylumbelliferyl phosphate (MUP, 1 mM, 3 ml) in each well of the 96-well plates are incubated for 60 min in the dark at room temperature. Fluorescence emits the product of the SEAP/MUP is measured at 449 nm using a 96-well plate fluorometer after excitation at 360 nm.

Macrophages RAW 264.7 are incubated with various concentrations of JSH-23 compound for 24 h. The cells are treated with WST-1 solution and absorbance is measured at 450 nm.(Only for Reference)